Enhanced protein production in higher plants by N-terminal fusion of a ubiquitin or a cucumber mosaic virus coat protein peptide

ABSTRACT

Methods are disclosed for enhancing protein production. One method comprises preparing a vector by inserting a gene encoding ubiquitin in front of a gene encoding a protein of interest and inserting the vector into a cell. A fusion protein will be expressed which includes ubiquitin plus the protein of interest. Ubiquitin C-terminal hydrolases can cleave the fusion protein leaving the desired protein in its free state. This method causes enhanced production of the protein of interest as compared to performing the same method without the ubiquitin gene as part of the vector. A ubiquitin promoter is unnecessary to yield this enhanced production and is not used. A second method is very similar except that in place of a ubiquitin gene, a gene encoding fourteen amino acids of cucumber mosaic virus coat protein is inserted in front of the gene of interest. This results in expression of a fusion protein comprising the fourteen amino acid residues of the coat protein bonded to the protein of interest. The fusion protein is produced at a higher level than is the protein when the coat protein gene fragment is not present in the vector. In both methods the genes can be placed under the control of heterologous promoters such as a 35S promoter.

BACKGROUND OF THE INVENTION

Strategies for production of proteins in heterologous fusion form havebeen widely applied in biotechnology for many purposes, such assecretion of proteins from host cells (fused to signal peptides), easydetection or purification of protein products (fused to reporter enzymesfor detection and to peptide tags for purification), searching forproteins with desired biological activities (e.g., in the phage displaytechnique and the two-hybrid system). Enhanced expression of proteins ofinterest has also been achieved by N-terminal fusion of a small peptideto the target protein. Fusion of a ubiquitin gene together with aubiquitin promoter to the 5′-end of a gene of interest is one of thesystems which has been used to enhance protein expression. Ubiquitinexists in all eukaryotic cells and is the most highly conserved proteinyet identified. It is abundant in cells and exhibits profound stabilityto heat and proteolytic degradation. Moreover, ubiquitin precursors,that is, polyubiquitin where ubiquitin monomers are linked up head totail and ubiquitin extension proteins where a single ubiquitin isappended at its C-terminus to either of two small ribosomal proteins,undergo rapid processing by ubiquitin C-terminal hydrolases, whichcleave C-terminal of the ubiquitin moieties and release the freeubiquitin monomer and the C-terminal extension proteins. All of thesefeatures have rendered ubiquitin as an excellent N-terminal fusionpartner to augment target protein accumulation in genetic engineering.

The ubiquitin fusion approach was first developed by Butt et al. (1989),who showed that fusion of ubiquitin to yeast metallothionein or to the αsubunit of the adenoylate cyclase-stimulatory GTP-binding proteinincreased the yield of these otherwise unstable or poorly expressedproteins from undetectable levels to 20% of the total cellular proteinsin E. coli. Ecker et al. (1989) demonstrated that in yeast, ubiquitinfusion resulted in enhanced expression of three mammalian proteins by upto 200-fold and all these ubiquitin fusion proteins were correctlyprocessed by yeast ubiquitin-specific endopeptidase to release authenticfunctional proteins. A similar yeast ubiquitin fusion expression systemwas reported by Sabin et al. (1989), in which ubiquitin/humanγ-interferon and ubiquitin/αl-proteinase inhibitor were highly expressedand quantitatively cleaved to yield γ-IFN and α1-PI with authentic aminotermini.

Since these early reports, a wealth of studies on ubiquitin fusionexpression of various proteins in E. coli and yeast have been described(Baker et al., 1994; Barr et al., 1991; Coggan et al., 1995; Gali andBoard, 1995; Gehring et al., 1995; Han et al., 1994; Kiefer et al.,1992; Lu et al., 1990; Lyttle et al., 1992; Mak et al., 1989; McDonnellet al., 1989; McDonnell et al., 1991; Pilon et al., 1996; Poletti etal., 1992; Rian et al., 1993; Tan and Board, 1996; Welch et al., 1995).Very often fusion to ubiquitin led to dramatic enhancement in yield ofthe fusion protein in bacteria, or of the cleaved product in yeast.

Enhanced expression of foreign proteins by ubiquitin fusion has alsobeen observed in plants. In analysis of the promoter of the tobaccopolyubiquitin gene, Ubi.U4, by driving transient expression of the GUSreporter in tobacco protoplasts, Genschik et al. (1994) found deletionof the intron sequence from the Ubi.U4 fragment spanning from −263 tothe end of the first ubiquitin-coding unit had no detectable influenceon the GUS activity, but further deletion of the ubiquitin-codingsequence diminished the GUS activity by 55%.

None of these studies has shown the direct enhancing function of theubiquitin fusion from a heterologous promoter. Garbarino and Belknap(1994) observed that fusion of the promoter plus ubiquitin-coding regionof the potato ubiquitin extension protein gene ubi 3 to the GUS reportergene resulted in GUS activity 5- to 10-fold higher than the directfusion of the ubi 3 promoter to the GUS gene did in transgenic potato.Again, the synergistic effect of the ubi 3 promoter and theubiquitin-coding sequence on the enhanced GUS activity was not excluded.In another study with a potato polyubiquitin gene, ubi 7, the same group(Garbarino et al., 1995) demonstrated that in transgenic potato plantsGUS expression level from the fusion construct containing the ubi 7promoter-5′ untranslated sequence-intron-first ubiquitin coding unit was10 times higher than that derived by only the ubi 7 promoter with the 5′untranslated sequence. However, the effects of the intron and theubiquitin protein fusion in increasing expression level of the GUSreporter were not clearly discriminated.

In addition to the above mentioned journal papers, a number of patentsrelated to the ubiquitin fusion technology have been filed since 1989.They are shown in Table 1. The publications and other materials usedherein to illuminate the background of the invention or provideadditional details respecting the practice, are incorporated byreference, and for convenience are respectively grouped in the appendedList of References.

TABLE 1 Patents related to the ubiquitin fusion technology Host TitleInventor Pat. No. Filing Date cells Generating desired amino- MIT WO8909829 Oct. 19, 1989 terminal residue in protein Regulation metabolicstability MIT US 5093242 Mar. 3, 1992 mammal, of a protein yeast Nucleicacid constructs, malaria Chiron WO 9208795 May 29, 1992 yeastpolypeptides and vaccines Production of a protein with a MIT US 5196321Mar. 23, 1993 E. coli predetermined amino-terminal amino acid residueYeast expression system for American EP 608532 Aug. 3, 1994 yeastretinoid-X receptor Cyanamid Recombinant DNA vectors Mascarenhas WO9423040 Oct. 13, 1994 E. coli New heat-inducible N-degron Varshavsky, WO9521269 Aug. 10, 1995 protein and nucleic acid Dohmen, encoding itJohnston, Wu Fusion proteins containing the Varshavsky, WO 9529195 Nov.2, 1995 N-or C-terminal of ubiquitin Johnston New fusion protein ofubiquitin Carbarino, WO 9603519 Feb. 8, 1996 plant plant and lyticpeptide Jaynes, Belknap Production of tissue factor Innis, WO 9604377Feb. 15, 1996 yeast pathway-inhibitor in yeast cells Creasey Stablerecombinant ubiquitin- J. Jaynes WO 9603522 Feb. 8, 1996 plant lyticpeptide fusion protein Fusion protein encoded by a Bachmair, US 5496721May 3, 1990 mammal, gene construct Finley, yeast Varshavsky

SUMMARY OF THE INVENTION

In accordance with the present invention a method for enhancingexpression of proteins in plants or plant cells is achieved by thefusion of a ubiquitin monomer coding sequence to the 5′ end of thecoding sequence of the proteins. Expression of the ubiquitin fusionproteins is driven by a promoter other than promoters from polyubiquitinprotein genes or ubiquitin extension protein genes. Thus enhancement ofexpression level of the proteins is due to the 5′ terminal addition ofthe ubiquitin monomer coding sequence. The ubiquitin fusion proteins arecleaved at the carboxy-terminal glycine 76 residue of the ubiquitin,presumably by plant ubiquitin specific proteases, to produce proteinswith desired biological properties. A second aspect of this invention isthat the N-terminal peptide of 14 amino acid residues of cucumber mosaicvirus coat protein (NP14) can be used as an N-terminal fusion partner toincrease the expression level of target proteins in plants. TheN-terminal fusion approaches described in this invention allow higheryield production of proteins in plants, either in the authentic forms inthe ubiquitin fusion system or as the fusion protein in the NP14 fusionsystem.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence and deduced amino acid sequence oftobacco ubi.NC89. The nucleotide sequence is listed as SEQ ID NO:1 andthe amino acid sequence is SEQ ID NO:2 in the Sequence Listing. Theprimers used in PCR are underlined and the mended 37-mer oligonucleotideis double-underlined.

FIG. 2 shows the synthetic DNA coding for the 14 N-terminal amino acidsof CMV CP (NP14). The nucleotide sequence is SEQ ID NO:3 and the aminoacid sequence is SEQ ID NO:4.

FIG. 3 illustrates the construction of the ubiquitin-GUS fusion proteinexpression vector pUG. The nucleotide sequence shown for pSKUBC1 is SEQID NO:5, the sequence shown for pBI122 is SEQ ID NO:6, and the sequenceshown for pUG is SEQ ID NO:7.

FIG. 4 illustrates the construction of the NP14-GUS fusion proteinexpression vector pCG. The nucleotide sequence shown for pUCG2 is SEQ IDNO:8.

FIG. 5 illustrates the construction of the ubiquitin-luciferase fusionprotein expression vector pUL. The arrow marked in the recognitionsequence of Stu I in pBIubi indicates the end of the ubiquitin codingregion and the cleavage site of the ubiquitin fusion protein. The uppernucleotide sequence shown for pBIubi is SEQ ID NO:9, the lowernucleotide sequence shown for pBIubi is SEQ ID NO:10, and the nucleotidesequence shown for pUL is SEQ ID NO:11.

FIG. 6 illustrates the construction of the NP14-luciferase fusionprotein expression vector. The nucleotide sequence shown for pCL is SEQID NO:12.

FIG. 7 illustrates the construction of ubiquitin-GUS fusion/LUC dualreport binary vector pUGL121.

FIG. 8 illustrates the construction of the NP14-GUS fusion/LUC dualreporter binary vector pCGL121.

FIG. 9 illustrates the construction of the GUS/LUC dual reporter binaryvector pBIL121.

FIG. 10 illustrates the ubiquitin fusion cloning vector pBIubi. Theupper nucleotide sequence is SEQ ID NO:13 and the lower nucleotidesequence is SEQ ID NO:14.

FIG. 11 illustrates the NP14 fusion cloning vector pBINP14.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods and constructs forenhancing protein production in plants. The methods comprise fusing anexpression-enhancing nucleic acid at the 5′ terminus of the gene forwhich enhanced expression is desired. In one aspect of the invention, aubiquitin gene is inserted in front of the gene encoding the desiredprotein such that a fusion protein is produced wherein ubiquitin isdirectly fused to the amino terminus of the desired protein. Enzymessuch as C-terminal hydrolases, will cleave at the C-terminus of theubiquitin in the fusion protein thereby releasing the desired protein inits natural form as well as forming free ubiquitin. The presence of theubiquitin gene in the resulting fusion protein results in enhancedexpression of the gene thereby yielding a greater amount of the desiredprotein product than occurs in the absence of the ubiquitin gene. It isnecessary to use only the coding portion of the ubiquitin gene. Theubiquitin promoter is unnecessary, and the ubiquitin gene fusion can beunder the control of a heterologous promoter.

In a second aspect of the invention, enhanced protein production is seenwhen a nucleic acid encoding 14 amino acids of cucumber mosaic viruscoat protein is placed in front of the gene encoding a desired proteinsuch that a fusion protein is produced wherein the fusion proteinincludes the 14 amino acids of the cucumber mosaic virus coat protein atthe amino terminus of the fusion protein.

The aspects of the invention are set out in the following Examples whichare offered by way of illustration and are not intended to limit theinvention in any manner. Standard techniques well known in the art orthe techniques specifically described below are utilized. Techniquessuch as transfection of protoplasts, preparation of transgenic tobaccoplants, fluorometric GUS assays and luciferase assays are well known tothose of skill in the art and are not described in detail herein.

EXAMPLE 1 DNA Sequences Coding for the Tobacco Ubiquitin and theN-terminal Peptide of CMV Coat Protein

The coding sequence of the ubiquitin monomer contains 228 base pairs.The 5′ part of 191 base pairs was obtained by polymerase chain reaction(PCR) amplification on the total DNA of Nicotiana tobacum var. NC89 andthe remaining 37 base pairs were prepared as a syntheticoligonucleotide. An SphI site encompassing the initiation codon ATG andan NcoI site following the last codon GGC were created to facilitatecloning. The tobacco ubiquitin coding sequence was then cloned intopGEM-5ZF and sequenced. FIG. 1 shows the DNA sequence and the deducedamino acid sequence of the tobacco ubiquitin. The 76-amino acid sequenceis identical to that derived from a tobacco polyubiquitin gene ubi.U4(Genschik et al., 1994). However, the nucleotide sequence of the regionamplified from the tobacco DNA is different from the correspondingregions of all ubiquitin monomers found in ubi.U4. We have named thistobacco ubiquitin coding sequence as ubi.NC89.

The cucumber mosaic virus coat protein (CMV CP) is encoded by the viralsubgenomic RNA 4 and comprises 218 amino acid residues. The CP gene ofthe strain CMV-SD was cloned by RT-PCR (Guo et al., 1993) and the cDNAsequence encoding the 14 N-terminal amino acids (NP14) was either cutout of the CP gene by NcoI/AccI digestion or chemically synthesized. Inthe synthesized version of the NP14 coding sequence (FIG. 2),overhanging adapters for BamIII and SstI sites were attached to the 5′-and 3′-ends, respectively, for easy cloning.

EXAMPLE 2 Translational Fusion Constructs for Transient ExpressionAssays

A. Ubiquitin-GUS Fusion Construct pUG

The ubi.NC89 sequence was taken from the plasmid pSKUBC1 as an XbaI-NcoI(filled-in) fragment and inserted into the XbaI-BamHI (filled-in) siteupstream of the GUS gene in pBI221 to construct pUG as shown in FIG. 3.

B. NP14-GUS Fusion Construct pCG

Plasmid pUCG2 is a derivative of pBI221, in which the ubi.NC89 sequenceand the NP14 sequence, linked as a read-through ORF, was inserted intothe XbaI-SmaI sites in front of the GUS gene. The ubiquitin moiety wasremoved from pUCG2 by XbaI-SacII digestion and pCG was formed byrecircularizing. FIG. 4 illustrates these steps clearly.

C. Ubiquitin-LUC Fusion Construct pUL

An NcoI (filled-in)-SstI fragment containing the firefly luciferase(LUC) gene was inserted into the ubiquitin fusion vector pBIubi (seeFIG. 10) downstream of ubi.NC89 via the StuI-SstI sites in thepolylinker region, resulting in pUL as shown in FIG. 5.

D. NP14-LUC Fusion Construct pCL

The NcoI (filled-in)-SstI fragment containing the LUC gene was insertedinto the NP14 fusion vector pBINP14 (see FIG. 11) downstream of the NP14coding sequence via AccI (or SalI which is the equivalent site here)(filled-in)-SstI sites, resulting in pCL as shown in FIG. 6.

EXAMPLE 3 GUS/LUC Dual Reporter Constructs for Stable Transformation

To examine the enhancing effects of the N-terminal addition of theubiquitin or CMV CP NP14 on GUS expression in stably transformed plants,a series of GUS/LUC (test/reference) dual reporter constructs were made.Essentially they are based on the fusion constructs used in transientexpression assays, namely, pUG and pCG. The chimeric GUS expressioncassettes were moved into the plant transformation intermediate plasmidpBI121, resulting in pUG121 and pCG121, respectively. The expressioncassette of the reference reporter LUC, which was constructed byreplacing the GUS gene in pBI221 with the LUC gene, was pre-made as aHindIII fragment (HindIII-35S/LUC/NOS-HindIII) and then inserted intothe unique HindIII site of pUG121, pCG121 and pBI121, respectively. Theresulting GUS/LUC dual reporter constructs, pUGL121, pCGL121 and pBIL121are shown in FIGS. 7, 8 and 9, respectively.

EXAMPLE 4 Ubiquitin Fusion Enhances the Expression of GUS and LUC inTobacco Protoplasts

The ubiquitin-GUS fusion construct pUG or the control plasmid pBI221 wasintroduced into tobacco protoplasts derived from tobacco BY-2 suspensioncells, together with a reference plasmid FFO which contained LUC genedriven by the 35S promoter. GUS activities were determined andnormalized by luciferase activities. In four independent transfectionexperiments, the normalized GUS activities (Δ GUS) from pUG wereconsiderably higher than those from pBI221. The averaged increase folddue to the ubiquitin fusion is 6.0 (Table 2). When using LUC as areporter and GUS as an internal standard as expressed from pBI221, thenormalized LUC activities from pUL were 1.37 to 3.11 fold higher thanthose from the control plasmid p35SLUC (35S-LUC-NOS) in threeindependent transfection experiments, with the average increase foldabout 2 (Table 3).

EXAMPLE 5 CMV CP NP14 is a More Efficient Fusion Partner than Ubiquitin

The enhancing effects of the NP14 fusion on GUS and LUC expression intobacco protoplasts were examined in experiments parallel to the abovementioned ubiquitin fusion study. The NP14-GUS fusion construct pCGproduced an average 11-fold higher GUS activity than did pBI221. Theseresults are shown in Table 2. Fusion of NP14 to LUC increased the LUCactivity by 2.87 times, calculated by comparing the normalized LUCactivity of pCL to that of p35SLUC. These results are shown in Table 3.It is apparent that NP14 is a more efficient fusion partner thanubiquitin in augmenting GUS and LUC expression in tobacco cells.

TABLE 2 Normalized GUS activities and enhancing fold of the N-terminalfusion constructs plasmid pBI221 pUG pCG activities GUS Δ GUS E Δ GUS E1 293.3 3760.0 12.8 5743.0 19.6 2 206.7 584.3 2.8 940.8 4.6 3 856.73733.8 4.4 6708.0 7.8 4 100.0 408.8 4.1 1247.0 12.5 average E value 6.0± 2.2 11.1 ± 3.2Notes: 1. The normalized GUS activity ΔGUS is calculated by the formula

${\Delta\;{GUS}_{n}} = \frac{{GUS}_{n} \times {LUC}_{221}}{{LUC}_{n}}$where n represents a particular GUS fusion construct, 221 representspBI221.

2. The enhancing fold E is calculated as

$\frac{\Delta\;{GUS}_{n}}{{GUS}_{221}}$

TABLE 3 Normalized LUC activities and enhancing fold of the N-terminalfusion constructs p35S LUC pUL pCL Plasmid average average averageactivities ΔLUC ΔLUC ΔLUC ΔLUC E ΔLUC ΔLUC E 1 1 252 290 274 396 1.37457 491 1.70 2 329 518 529 2 1 169 169 556 526 3.11 701 794 4.70 2 ND496 886 3 1  64 112 141 164 1.46 270 246 2.20 2 160 181 254 3 ND 170 214Mean ± SE 1.98 ± 0.56 2.87 ± 0.92Notes: 1. The normalized LUC activity ΔLUC is calculated by the formula

${\Delta\;{LUC}_{n}} = \frac{{{LUC}_{n} \times {GUSp}}\; 35{SLUC}}{{GUS}_{n}}$

where n represents a particular LUC fusion construct.

2. The enhancing fold E is calculated as

$\frac{\Delta\;{LUC}_{n}}{{LUCp}\; 35{SLUC}}.$

EXAMPLE 6 Ubiquitin- and NP14-Fusion Enhance GUS Expression inTransgenic Plants

To examine the enhancing effects of the ubiquitin fusion and the NP14fusion on GUS expression in stably transformed plants, three GUS/LUC(test/reference) dual reporter constructs were made based on the binaryvector pBI121. pUGL121, pCGL121 and pBIL121 contained expressioncassettes ubiquitin-GUS, NP14-GUS and GUS only (control), respectively,and the reference LUC expression cassette was integrated in each plasmid(FIGS. 7–9). Tobacco plants transformed with each of the threeconstructs were prepared and analyzed for GUS and LUC activities. Eachplant was analyzed twice in two independent experiments and only thoseplants displaying reasonable consistency of the relative GUS activities(GUS/LUC) in two experiments % were included for comparison. As shown inTable 4, although variations in the relative GUS activities existedamong different transformants from the same constructs, the average GUSexpression level of 5 qualified plants containing the 35S-ubiquitin/GUSfusion construct was 4 times higher than that derived from 6 plantscontaining the 35S-GUS construct, confirming the enhancing effect of theubiquitin fusion on GUS expression as previously observed in tobaccoprotoplasts. Again, the NP14 fusion displayed a higher enhancing effecton GUS expression than did the ubiquitin fusion. The average relativeGUS activity of 14 pCGL plants was about 7 fold that derived from thepBIL121 construct.

EXAMPLE 7 Ubiquitin Fusion and NP14 Fusion Cloning Vectors

pBIubi (FIG. 10) and pBINP14 (FIG. 11) are two fusion protein expressionvectors allowing for insertion of target genes downstream of theubi.NC89 and the CMV CP NP14 coding sequence, respectively. Both vectorsare derivatives of pBI221, with the GUS gene being replaced by theubi.NC89 or NP14 coding sequence. In pBIubi, a polylinker sequence wasattached to the 3′ end of the ubi.NC89 sequence and the penultimatecodon of the ubi.NC89 was changed from GGT to GGA for creating a StuIsite in the polylinker region. In pBINP14, two cloning sites SalI (hereequivalent to an AccI site) and SstI, are available for cloning thetarget genes downstream from the NP14 sequence (the last 5 base pairs ofthe NP14 sequence form part of the SalI recognition sequence). In orderto use AccI instead of SalI for cleaving pBINP14, the AccI site at −393of the CaMV 35S promoter was eliminated.

TABLE 4 Effects of ubiquitin- and NP14-fusion on GUS expression intrangenic tobacco plants Relative GUS activities: GUS/LUC (pmol MUmin⁻¹/cpm × 10⁻³) pUGL121 pCGL121 pBIL121 Plant lines exp. 1 exp. 2average exp. 1 exp. 2 average exp. 1 exp. 2 average  1 12.9 15.3 14.12.4 3.4 2.9 1.4 2.6 2  2 13 43 28 4.5 6.8 5.65 5.2 2.4 3.8  3 0.7 0.50.6 63.2 9.5 36.35 4.2 0.6 2.4  4 0.3 0.4 0.35 26.9 8.3 17.6 2.5 5.43.95  5 4.8 0.8 2.8 17.8 22.2 20 0.4 0.38 0.39  6 2.1 5 3.55 0.5 0.820.66  7 4.6 5.8 5.2  8 58.7 20.2 39.45  9 15.6 3.6 9.6 10 17.2 4.4 10.811 3 1.4 2.2 12 17.9 24.2 21.05 13 20.7 19.4 20.05 14 13.7 25.3 19.5Mean ± SE 9.17 ± 5.34 15.28 ± 3.18 2.2 ± 0.61

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than in a limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, within the spirit of the invention and thescope of the appended claims.

1. A method for enhancing production of a desired protein as part of a fusion protein in a plant cell or a plant which method comprises providing a nucleic acid which encodes a fusion protein to a plant cell or plant, wherein the fusion protein comprises the protein of SEQ ID NO: 4 linked to the desired protein and wherein the desired protein is heterologous to the protein of SEQ ID NO:4.
 2. The method of claim 1 wherein the carboxy terminus of said protein of SEQ ID NO:4 forms a peptide linkage with the amino terminus of said desired protein.
 3. The method of claim 1 wherein said nucleic acid comprises nucleotides 6–47 of SEQ ID NO:3.
 4. The method of claim 1 wherein said nucleic acid is under the control of a CaMV 35S promoter.
 5. A nucleic acid vector comprising a plant-expressible promoter operably linked to a nucleic acid which encodes a fusion protein wherein said fusion protein comprises the protein of SEQ ID NO:4 linked to a protein of interest, wherein the protein of interest is heterologous to the protein of SEQ ID NO:4.
 6. The vector of claim 5 wherein said protein of SEQ ID NO:4 is linked in a peptide linkage at its carboxy terminus to the amino terminus of said protein of interest.
 7. The vector of claim 5 wherein said nucleic acid is under the control of a CaMV 35S promoter.
 8. The vector of claim 5 wherein said vector comprises nucleotides 6–47 of SEQ ID NO:3.
 9. A plant cell or a plant comprising the vector of claim
 5. 10. An isolated nucleic acid comprising SEQ ID NO:3.
 11. An isolated nucleic acid consisting of SEQ ID NO:3.
 12. The method of claim 1, wherein the plant cell or plant is transfected with the nucleic acid.
 13. The method of claim 1, wherein the plant cell or plant is transformed with the nucleic acid.
 14. A plant cell or a plant comprising the vector of claim
 6. 15. A plant cell or a plant comprising the vector of claim
 7. 